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While new biomaterials for regenerative therapies are being reported in the literature, clinical translation is slow. Some existing regenerative approaches rely on high doses of growth factors, such as bone morphogenetic protein‐2 (BMP‐2) in bone regeneration, which can cause serious side effects. An ultralow‐dose growth factor technology is described yielding high bioactivity based on a simple polymer, poly(ethyl acrylate) (PEA), and mechanisms to drive stem cell differentiation and bone regeneration in a critical‐sized murine defect model with translation to a clinical veterinary setting are reported. This material‐based technology triggers spontaneous fibronectin organization and stimulates growth factor signalling, enabling synergistic integrin and BMP‐2 receptor activation in mesenchymal stem cells. To translate this technology, plasma‐polymerized PEA is used on 2D and 3D substrates to enhance cell signalling in vitro, showing the complete healing of a critical‐sized bone injury in mice in vivo. Efficacy is demonstrated in a Münsterländer dog with a nonhealing humerus fracture, establishing the clinical translation of advanced ultralow‐dose growth factor treatment.

INTRODUCTION During recent years, microgels have emerged as an effective type of drug delivery system (DDS), showing advantages such as tuneable size, increased surface area and injectability1. Collagen has been extensively studied as a scaffold for regenerative medicine and drug delivery due to its biocompatibility, non-immunogenicity and degradability2,3. The current study focuses on the use of microfluidic techniques for the automated generation of monodisperse type-I collagen (col-I) microgels crosslinked with PEG-4S, encapsulating hollow collagen spheres with glial-derived neurotrophic factor (GDNF) and bone morphogenetic protein 2 (BMP-2) for regenerative therapies in Parkinson’s Disease and bone repair, respectively. MATERIALS AND METHODS Col-1 (Collagen Solutions, UK) stock was characterized in a rotational viscometer and UV-Vis spectrophotometer to determine the fibrillogenesis conditions. Collagen was crosslinked with 4S-Star-PEG succinimide glutarate (Jenkem, USA) to form hydrogels. Crosslinking occurred after merging col-I with PEG-4S crosslinker in the nozzle of a double-chamber capillary within an oil flow. A coiled tube was placed at the outlet for microgels to gelify inside the device. Microgel size was controlled varying the nozzle diameter and flow speeds, and the mechanical properties modulated through pH and concentration of collagen/crosslinker. Microgels were characterized with TNBS assay and their cytotoxicity with LIVE-DEAD® assay (Neu-7 astrocytes, n=3). Hollow spheres were prepared by covalently attaching collagen to silica templates that are later dissolved with hydrofluoric acid. They were characterized morphologically and chemically, namely: scanning electron microscopy, dynamic light scattering and Fourier-Transform Infrared (FTIR). Hollow spheres were loaded with GDNF and BMP-2 by diffusion, seeded with different cell-types and encapsulated in the microgels within the microfluidic device. RESULTS AND DISCUSSION Microgels with different size and stiffness were successfully synthetized in a glass microfluidic device. The microgels are non-cytotoxic to cells (fig.1C) and foster cell growth at different crosslinker concentrations. In the synthesis of hollow spheres, no harmful by-products appeared (fig.1D), and their size ranged around 200 nm (fig.1E). Microfluidic-generated hydrogels encapsulating hollow spheres can therefore be used for sustained delivery of GDNF and BMP-2. CONCLUSIONS We demonstrate that microfluidics is an adequate technique for automatically generating monodisperse collagen microgels and provides a simple and useful tool for the posterior encapsulation of nanospheres and cells. The combination of microgels encapsulating hollow spheres is expected to provide a controlled and sustained delivery system of different therapeutic factors. AKNOWLEDGEMENTS The authors would like to thank European Union H2020 Programme (H2020-MSCA-ITN-2015) and Grant No. 676408 for providing financial support to this project.

Silanization has emerged in recent years as a way to obtain a stronger and more stable attachment of biomolecules to metallic substrates. However, its impact on protein conformation, a key aspect that influences cell response, has hardly been studied. In this work, we analyzed by atomic force microscopy (AFM) the distribution and conformation of type I collagen on plasma-treated surfaces before and after silanization. Subsequently, we investigated the effect of the different collagen conformations on fibroblasts adhesion and fibronectin secretion by immunofluorescence analyses. Two different organosilanes were used on plasma-treated titanium surfaces, either 3-chloropropyl-triethoxy-silane (CPTES) or 3-glycidyloxypropyl-triethoxy-silane (GPTES). The properties and amount of the adsorbed collagen were assessed by contact angle, X-ray photoelectron spectroscopy, optical waveguide lightmode spectroscopy, and AFM. AFM studies revealed different conformations of type I collagen depending on the silane employed. Collagen was organized in fibrillar networks over very hydrophilic (plasma treated titanium) or hydrophobic (silanized with CPTES) surfaces, the latter forming little globules with a beads-on-a-string appearance, whereas over surfaces presenting an intermediate hydrophobic character (silanized with GPTES), collagen was organized into clusters with a size increasing at higher protein concentration in solution. Cell response was strongly affected by collagen conformation, especially at low collagen density. The samples exhibiting collagen organized in globular clusters (GPTES-functionalized samples) favored a faster and better fibroblast adhesion as well as better cell spreading, focal adhesions formation, and more pronounced fibronectin fibrillogenesis. In contrast, when a certain protein concentration was reached at the material surface, the effect of collagen conformation was masked, and similar fibroblast response was observed in all samples.

INTRODUCTION During recent years, microgels have emerged as an effective type of drug delivery system (DDS), showing advantages such as tuneable size, increased surface area and injectability1. Collagen has been extensively studied as a scaffold for regenerative medicine and drug delivery due to its biocompatibility, non-immunogenicity and degradability2,3. The current study focuses on the use of microfluidic techniques for the automated generation of monodisperse type-I collagen (col-I) microgels crosslinked with PEG-4S, encapsulating hollow collagen spheres with glial-derived neurotrophic factor (GDNF) and bone morphogenetic protein 2 (BMP-2) for regenerative therapies in Parkinson’s Disease and bone repair, respectively. MATERIALS AND METHODS Col-1 (Collagen Solutions, UK) stock was characterized in a rotational viscometer and UV-Vis spectrophotometer to determine the fibrillogenesis conditions. Collagen was crosslinked with 4S-Star-PEG succinimide glutarate (Jenkem, USA) to form hydrogels. Crosslinking occurred after merging col-I with PEG-4S crosslinker in the nozzle of a double-chamber capillary within an oil flow. A coiled tube was placed at the outlet for microgels to gelify inside the device. Microgel size was controlled varying the nozzle diameter and flow speeds, and the mechanical properties modulated through pH and concentration of collagen/crosslinker. Microgels were characterized with TNBS assay and their cytotoxicity with LIVE-DEAD® assay (Neu-7 astrocytes, n=3). Hollow spheres were prepared by covalently attaching collagen to silica templates that are later dissolved with hydrofluoric acid. They were characterized morphologically and chemically, namely: scanning electron microscopy, dynamic light scattering and Fourier-Transform Infrared (FTIR). Hollow spheres were loaded with GDNF and BMP-2 by diffusion, seeded with different cell-types and encapsulated in the microgels within the microfluidic device. RESULTS AND DISCUSSION Microgels with different size and stiffness were successfully synthetized in a glass microfluidic device. The microgels are non-cytotoxic to cells (fig.1C) and foster cell growth at different crosslinker concentrations. In the synthesis of hollow spheres, no harmful by-products appeared (fig.1D), and their size ranged around 200 nm (fig.1E). Microfluidic-generated hydrogels encapsulating hollow spheres can therefore be used for sustained delivery of GDNF and BMP-2. CONCLUSIONS We demonstrate that microfluidics is an adequate technique for automatically generating monodisperse collagen microgels and provides a simple and useful tool for the posterior encapsulation of nanospheres and cells. The combination of microgels encapsulating hollow spheres is expected to provide a controlled and sustained delivery system of different therapeutic factors. AKNOWLEDGEMENTS The authors would like to thank European Union H2020 Programme (H2020-MSCA-ITN-2015) and Grant No. 676408 for providing financial support to this project., {"references":["Oh JK, Drumright R, Siegwart DJ, Matyjaszewski K. The development of microgels/nanogels for drug delivery applications. Prog Polym Sci. 2008 Apr 1;33(4):448–77.","Lee CH, Singla A, Lee Y. Biomedical applications of collagen. Int J Pharm. 2001 Jun 19;221(1):1–22.","An B, Lin Y-S, Brodsky B. Collagen interactions: Drug design and delivery. Extracell Matrix ECM ECM- Mater Ther Tools Targets Cancer Treat. 2016 Feb 1;97:69–84.","Gasperini L, Mano JF, Reis RL. Natural polymers for the microencapsulation of cells. J R Soc Interface. 2014 Nov 6;11(100):20140817.","Fontana G, Srivastava A, Thomas D, Lalor P, Dockery P, Pandit A. Three-Dimensional Microgel Platform for the Production of Cell Factories Tailored for the Nucleus Pulposus. Bioconjug Chem. 2015 Jul 15;26(7):1297–306."]}